Proteomic analysis of Daphnia magna hints at molecular pathways involved in defensive plastic responses

Abstract

Background: Phenotypic plasticity in defensive traits occurs in many species when facing heterogeneous predator regimes. The waterflea Daphnia is well-known for showing a variety of these so called inducible defences. However, molecular mechanisms underlying this plasticity are poorly understood so far. We performed proteomic analysis on Daphnia magna exposed to chemical cues of the predator Triops cancriformis. D. magna develops an array of morphological changes in the presence of Triops including changes of carapace morphology and cuticle hardening.

Results: Using the 2D-DIGE technique, 1500 protein spots could be matched and quantified. We discovered 179 protein spots with altered intensity when comparing Triops exposed animals to a control group, and 69 spots were identified using nano-LC MS/MS. Kairomone exposure increased the intensity of spots containing muscle proteins, cuticle proteins and chitin-modifying enzymes as well as enzymes of carbohydrate and energy metabolism. The yolk precursor protein vitellogenin decreased in abundance in 41 of 43 spots.

Conclusion: Identified proteins may be either directly involved in carapace stability or reflect changes in energy demand and allocation costs in animals exposed to predator kairomones. Our results present promising candidate proteins involved in the expression of inducible defences in Daphnia and enable further in depth analysis of this phenomenon.

Figure 1

Inducible defence in D. magna. Adult D. magna showing increased bulkiness after Triops induction (right) compared to control animal (left). (Photo: M. Rabus).

Figure 2

2D DIGE gel for comparing Triops exposed and control D. magna embryos. Spots with more abundant proteins in the kairomone exposed group are displayed in red (Cy5 labelled), spots with more abundant proteins in the control group are displayed in green (Cy3 labelled). Spots marked with Spot ID showed significantly different intensity and were successfully identified. Spot IDs not listed in Table 1 or Table 2 refer to vitellogenin-related spots.

Figure 3

Examples for normalised DIGE intensity ratios. Normalisation was done according to internal pooled standard (IPS), here an abundance of e.g. 2 indicates that abundance is 2 of IPS abundance whereas -2 means 1/2 of IPS abundance. They serve as indicators for changes in protein abundance in kairomone exposed D. magna and in the control group for: Spot 331 – STAT Protein (A); Spot 619 – Chitin deacetylase 2A (B); Spot 1517 – Vitellogenin (C) and Spot 1929 – Cuticle Protein (D).

Figure 4

Hierarchical clustering heat map of all protein spots present in at least two biological replicates. Graph was created with R using the heatmap.2 function of package gplots. Rows indicate proteins whereas columns represent biological replicates of Triops kairomone exposed animals (triops) and control group (control).

Figure 5

Annotation term network created with ClueGo using functional annotation analysis (two-sided hypergeometric test, Benjamini-Hochberg-correction, kappa-score ≥0.3). FlybaseIDs of proteins with increased and decreased abundance were searched against GO and KEGG databases. Small circles show involved genes and large circles refer to GO terms. Arrows next to gene names indicate decreased or increased abundance. Colours represent grouping of GO terms whereas size of circle and circle label illustrate the corrected p-value. Abbreviated Drosophila gene names correspond to the following protein names (compare also Tables 1 and 2): Ald – Fructose-Bisphosphate aldolase, ATPsyn-beta – ATP synthase beta, awd – Nucleoside diphosphate kinase, CG10688 – Phosphomannomutase, Cpr50Cb – Cuticle protein, Cpr65Ax1 – Cuticle protein 1c, Edg78E – Cuticle protein 1b, Gapdh2 – GAPDH, l(2)37Cc – Prohibitin, Prm – Paramyosin, serp – Chitin deacetylase 1, verm – Chitin deacetylase 2A, wupA – Troponin.

Figure 6

Setup of induction experiment. One replicate consists of one beaker with daphnids and a net cage containing the predator, so that daphnids perceive chemical cues of Triops but were prevented from being eaten. For the control group, the net cage was empty.